Members of the Mycobacterium avium complex (MAC) are a family of intracellular bacterial pathogens causing significant disease in both animals and humans. The complex contains four subspecies of M. avium: M. avium subsp. avium (MAA), M. avium subsp. paratuberculosis (MAP), M. avium subsp. hominissuis (MAH), and M. avium subsp. silvaticum (MAS). Mycobacterium intracellulare is also a member of the complex.
The most common method for diagnosis of mycobacterial infections in humans and animals begins with growth (isolation) of the causative organism in liquid culture media. While direct detection technology (such as PCR) can be done on clinical samples, most studies find trying to find the bacteria's DNA less sensitive than culture-based methods. Also, culture yields the pathogens needed for subsequent testing such as antibiotic susceptibility or molecular epidemiology. The success of culture-based methods for mycobacteria depends heavily on specimen processing techniques that eliminate non-mycobacterial microflora from the sample. The tubes of inoculated liquid media are monitored by instruments that incubate and “examine” the culture for evidence of microbial growth using indicators such as oxygen consumption or gas pressure inside the sealed culture vessel. Examples of these diagnostic systems are the BACTEC MGIT 960 system (BD Diagnostic Systems, Franklin Lakes, N.J.), and Trek ESP II system (Trek Diagnostic Systems, Cleveland, Ohio). These instruments generally handle 300-1,000 cultures at a time and cost in excess of $50,000. The culture media associated with each instrument retails for $5 to $10 per sample tube depending on instrument lease agreements and volume discounts.
After an instrument signals that microbial growth is occurring in the culture tube, a variety of assays must be performed to identify the micro-organism(s) in the tube that triggered the signal. Such assays vary in sensitivity, specificity, and cost. Possible outcomes of mycobacteria identification assays on isolates from liquid cultures include: 1) false-signal by the instrument, i.e. no microorganisms were detected, 2) identification of a mycobacterial pathogen of limited clinical significance, e.g. a non-pathogenic environmental Mycobacterium sp., or 3) identification of a mycobacterial pathogen of importance.
The vast majority of clinically important mycobacterial pathogens generally fall into two groups: Mycobacterium tuberculosis Complex (MTBC) and the Mycobacterium avium complex (MAC). Mycobacterium leprae, the cause of leprosy, is also an important mycobacterial pathogen but it is far less common and is presently not cultivable in vitro. In developing countries MTBC is the predominant mycobacterial pathogen of concern. In developed countries MAC pathogens are more common, particularly in immunocompromised patients such as people with HIV or under treatment for cancer.
Typically, definitive identification of mycobacterial pathogens isolated from culture is primarily based on PCR. The PCR assays differ in design from those used for direct pathogen detection in clinical samples and are marketed specifically for mycobacterial identification from cultures. An example of one of the leading products in this market is AccuProbe® (Gen-Probe, San Diego, Calif.). Most such assays are target pathogen DNA-specific and rule in/out a specific pathogen group, e.g. MTBC or MAC. If the assay is positive, the result is reported as, for example, MAC complex positive. The specific species of mycobacteria within the complex is usually not determined. If the PCR assay is negative, other PCRs may be required to arrive at a diagnosis. The PCR assays may be costly and laborious.
A positive signal appears in automated liquid culture systems used for mycobacteria isolation when growth of a microorganism triggers the system's sensor. Rapid, low-cost methods are therefore desired to weed out diagnostically irrelevant cultures that can be signal-positive due to non-mycobacterial organisms. The present invention provides compositions and methods for achieving these and related objectives.